Mouse cell line authentication

Abstract

A multiplex polymerase chain reaction assay that targets nine tetranucleotide short tandem repeat (STR) markers in the mouse genome. Unique profiles were obtained from seventy-two mouse samples that were used to determine the allele distribution for each STR marker. Correlations between allele fragment length and repeat number were determined with DNA Sanger sequencing. Genotypes for L929 and NIH3T3 cell lines were shown to be stable with increasing passage numbers as there were no significant differences in fragment length with samples of low passage when compared to high passage samples. In order to detect cell line contaminants, primers for two human STR markers were incorporated into the multiplex assay to facilitate detection of human and African green monkey DNA. This multiplex assay is the first of its kind to provide a unique STR profile for each individual mouse sample and can be used to authenticate mouse cell lines.

We claim: 
     
       1. A method of determining the alleles present in a DNA sample, the method comprising:
 obtaining a DNA sample to be analyzed; 
 selecting a set of STR marker loci of the DNA sample to be analyzed that can be amplified together in a multiplex amplification reaction, wherein the set of STR marker loci are selected from the group consisting of: 18-3, 4-2, 6-7, 9-2, 15-3, 6-4, 12-1, 5-5 and X-1; 
 providing a set of oligonucleotide primer pairs, wherein each oligonucleotide primer pair in the set flanks a single locus in the set of STR marker loci, and wherein each oligonucleotide primer pair is capable of amplification of a single locus from the set of STR marker loci in a multiplex amplification reaction; 
 co-amplifying the set of STR marker loci in a multiplex amplification reaction, wherein the product of the multiplex amplification reaction comprises a mixture of amplified alleles from each of the co-amplified loci in the set of STR marker loci; 
 evaluating the products of the co-amplification reaction to determine the alleles present at each of the loci analyzed in the set of STR marker loci within the DNA sample; and 
 wherein the source of the DNA sample to be analyzed is at least one of a mouse and a cell line derived from a mouse. 
 
     
     
       2. The method of  claim 1 , wherein the mouse is selected from the group consisting of Mus musculus musculus, Mus musculus domesticus, Mus musculus molossinus and Mus musculus castaneus. 
     
     
       3. The method of  claim 1 , wherein the DNA sample to be analyzed comprises DNA derived from at least two different mouse sources. 
     
     
       4. The method of  claim 1 , wherein the DNA sample to be analyzed further comprises DNA derived from at least one other source that is a non-mouse source. 
     
     
       5. The method of  claim 4 , wherein the non-mouse source is at least one of human and African green monkey. 
     
     
       6. The method of  claim 5 , wherein the set of oligonucleotide primer pairs further comprises an oligonucleotide primer pair configured to amplify an STR allele from the non-mouse source. 
     
     
       7. The method of  claim 1 , wherein at least one oligonucleotide primer in each oligonucleotide pair comprises a detection label. 
     
     
       8. The method of  claim 7 , wherein the detection label comprises a fluorescent dye. 
     
     
       9. The method of  claim 8 , wherein evaluating the products of the co-amplification reaction comprises measuring fluorescence emission upon excitation of the detection label with light. 
     
     
       10. The method of  claim 7 , comprising using at least four oligonucleotide primer pairs, wherein at least one primer of each oligonucleotide primer pair is labeled with a detection label, and wherein at least four different detection labels are used.